This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microsporidia are obligately intracellular, single-celled fungal parasites that infect invertebrate and vertebrate hosts. Infections due to microsporidia occur worldwide in both immune-deficient and immune-competent humans of all age groups and are associated with diarrhea and systemic disease. Molecular-based PCR methods have improved sensitivity and specificity for diagnosing microsporidiosis but require costly equipment (eg. thermocycler) and take hours to perform. In this study, an accelerated LAMP method was applied to improve the time efficiency for molecular detection of the two most prevalent microsporidia species infecting humans, Enterocytozoon bieneusi and Encephalitozoon intestinalis. Primer Explorer LAMP software (http://primerexplorer.jp/e/v3_manual/index.html) was used to select primers from the ribosomal RNA gene targets of Ent. bieneusi (Genbank L07123) and Enc. intestinalis (Genbank L19567). Control Ent. bieneusi and Enc. intestinalis spores were obtained from rhesus macaque bile and tissue culture supernatants, respectively, and reacted with 0.8 uM FIP and BIP primers, 0.2 uM F3 and B3 primers, 0.4 uM LF and LB loop primers, 1.5 M betaine, 16 units Bst polymerase, and 0.5 M dNTPs in Bst buffer at a final volume of 50 ul for 1 hour at 63[unreadable] C followed by 10 min enzyme deactivation at 80[unreadable] C. Lower concentrations of betaine or higher concentrations of dNTPs typically generated negative results. The advantages of the accelerated LAMP over standard and nested PCR include lower cost, shorter time, higher specificity, and no requirement for special equipment. Accelerated LAMP, however, was approximately 10-fold less sensitive than nested PCR. These results support continued work to improve the sensitivity of accelerated LAMP to more efficiently detect microsporidia-positive specimens in clinical laboratory and field studies.